Identification of an intracellular peptide segment involved in sodium channel inactivation

Antibodies directed against a conserved intracellular segment of the sodium channel alpha subunit slow the inactivation of sodium channels in rat muscle cells. Of four site-directed antibodies tested, only antibodies against the short intracellular segment between homologous transmembrane domains III and IV slowed inactivation, and their effects were blocked by the corresponding peptide antigen. No effects on the voltage dependence of sodium channel activation or of steady-state inactivation were observed, but the rate of onset of the antibody effect and the extent of slowing of inactivation were voltage-dependent. Antibody binding was more rapid at negative potentials, at which sodium channels are not inactivated; antibody-induced slowing of inactivation was greater during depolarizations to more positive membrane potentials. The peptide segment recognized by this antibody appears to participate directly in rapid sodium channel inactivation during large depolarizations and to undergo a conformational change that reduces its accessibility to antibodies as the channel inactivates.     

Reorganization of the axon membrane in demyelinated peripheral nerve fibers: morphological evidence

Cytochemical staining of demyelinated peripheral axons revealed two types of axon membrane organization, one of which suggests that the demyelinated axolemma acquires a high density of sodium channels. Ferric ion-ferrocyanide stain was confined to a restricted region of axon membrane at the beginning of a demyelinated segment or was distributed throughout the demyelinated segment of axon. The latter pattern represents one possible morphological correlate of continuous conduction through a demyelinated segment and suggests a reorganization of the axolemma after demyelination.     

Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits

Axons in the cerebral cortex receive synaptic input at the axon initial segment almost exclusively from gamma-aminobutyric acid-releasing (GABAergic) axo-axonic cells (AACs). The axon has the lowest threshold for action potential generation in neurons; thus, AACs are considered to be strategically placed inhibitory neurons controlling neuronal output. However, we found that AACs can depolarize pyramidal cells and can initiate stereotyped series of synaptic events in rat and human cortical networks because of a depolarized reversal potential for axonal relative to perisomatic GABAergic inputs. Excitation and signal propagation initiated by AACs is supported by the absence of the potassium chloride cotransporter 2 in the axon.     

A conserved domain in axonal targeting of Kv1 (Shaker) voltage-gated potassium channels

Axonal voltage-gated potassium (Kv1) channels regulate action-potential invasion and hence transmitter release. Although evolutionarily conserved, what mediates their axonal targeting is not known. We found that Kv1 axonal targeting required its T1 tetramerization domain. When fused to unpolarized CD4 or dendritic transferrin receptor, T1 promoted their axonal surface expression. Moreover, T1 mutations eliminating Kvbeta association compromised axonal targeting, but not surface expression, of CD4-T1 fusion proteins. Thus, proper association of Kvbeta with the Kv1 T1 domain is essential for axonal targeting.     

Functional differences between two classes of sodium channels in developing rat skeletal muscle

Excitability is generated in developing skeletal muscle by the incorporation of sodium-selective ion channels into the surface membrane. Whole-cell and patch voltage-clamp recording from myotubes and their embryologic precursors, myoblasts, indicated that voltage-activated sodium current in myoblasts was more resistant to block by tetrodotoxin (TTX) than that in myotubes. Single-channel recording from both cell types showed two classes of sodium channels. One class had a lower single-channel conductance, activated at more hyperpolarized voltages, and was more resistant to TTX than the other. The proportion of TTX-resistant to TTX-sensitive sodium channels was higher in myoblasts than in myotubes. Thus, the difference in TTX sensitivity between myoblasts and myotubes can be explained by a difference in the proportion of the two classes of sodium channels. In addition, the lower conductance of TTX-resistant channels provides insight into the relationship between the TTX binding site and the external mouth of the sodium channel.     

Functional properties of rat brain sodium channels expressed in a somatic cell line

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Segregation of axonal and somatic activity during fast network oscillations

In central neurons, information flows from the dendritic surface toward the axon terminals. We found that during in vitro gamma oscillations, ectopic action potentials are generated at high frequency in the distal axon of pyramidal cells (PCs) but do not invade the soma. At the same time, axo-axonic cells (AACs) discharged at a high rate and tonically inhibited the axon initial segment, which can be instrumental in preventing ectopic action potential back-propagation. We found that activation of a single AAC substantially lowered soma invasion by antidromic action potential in postsynaptic PCs. In contrast, activation of soma-inhibiting basket cells had no significant impact. These results demonstrate that AACs can separate axonal from somatic activity and maintain the functional polarization of cortical PCs during network oscillations.     

ThMYB3和ThDof2对ThVHAc1基因表达的调控

ThVHAc1基因能响应干旱、盐、重金属等胁迫诱导,该基因能有效提高转ThVHAc1基因酵母的多种抗逆能力。为进一步研究ThVHAc1基因的抗逆调控机理,本研究利用酵母单杂交技术钓取ThVHAc1可能的上游调控因子,并利用基因枪瞬时共转化技术进行初步验证。酵母单杂交结果显示,ThMYB3基因能识别ThVHAc1基因上游MYB顺式作用元件和含有MYB元件的启动子片段。ThDof2基因能识别ThVHAc1基因上游DOF顺式作用元件和含有DOF元件的启动子片段。但ThMYB3和ThDof2均不能识别突变后的元件及含对应突变元件的启动子片段。将ThMYB3和ThDof2分别构建成效应载体,各元件、突变元件、启动子片段及突变的启动子片段分别构建报告载体进行共表达验证。结果发现:效应载体ThMYB3和含MYB元件及含MYB元件的ThVHAc1启动子片段的报告载体,ThDof2和含DOF元件及含DOF元件的ThVHAc1启动子片段的报告载体瞬时共表达烟草叶片能观察到GUS染色,且GUS活性较高;而与相应突变元件及片段的共转化则观察不到烟草叶片的GUS染色,且GUS活性很低。说明只有非突变的元件和启动子片段才能与效应载体进行互作识别,激活GUS基因的表达,验证了酵母单杂交获得的上游调控基因的识别特异性。表明ThMYB3和ThDof2可能通过与相应启动子元件的结合调控ThVHAc1基因。      

Beta-adrenergic inhibition of cardiac sodium channels by dual G-protein pathways

The signaling pathways by which beta-adrenergic agonists modulate voltage-dependent cardiac sodium currents are unknown, although it is likely that adenosine 3'5'-monophosphate (cAMP) is involved. Single-channel and whole-cell sodium currents were measured in cardiac myocytes and the signal transducing G protein Gs was found to couple beta-adrenergic receptors to sodium channels by both cytoplasmic (indirect) and membrane-delimited (direct) pathways. Hence, Gs can act on at least three effectors in the heart: sodium channels, calcium channels, and adenylyl cyclase. The effect on sodium currents was inhibitory and was enhanced by membrane depolarization. During myocardial ischemia the sodium currents of depolarized cells may be further inhibited by the accompanying increase in catecholamine levels.     

The cell and molecular basis of mechanical, cold, and inflammatory pain

Peripheral pain pathways are activated by a range of stimuli. We used diphtheria toxin to kill all mouse postmitotic sensory neurons expressing the sodium channel Nav1.8. Mice showed normal motor activity and low-threshold mechanical and acute noxious heat responses but did not respond to noxious mechanical pressure or cold. They also showed a loss of enhanced pain responses and spontaneous pain behavior upon treatment with inflammatory insults. In contrast, nerve injury led to heightened pain sensitivity to thermal and mechanical stimuli indistinguishable from that seen with normal littermates. Pain behavior correlates well with central input from sensory neurons measured electrophysiologically in vivo. These data demonstrate that Na(v)1.8-expressing neurons are essential for mechanical, cold, and inflammatory pain but not for neuropathic pain or heat sensing.     

Two molecular transitions influence cardiac sodium channel gating

Sodium channels from diverse excitable membranes are very similar in their structure, yet surprisingly heterogeneous in their behavior. The processes that govern the opening and closing of sodium channels have appeared difficult to describe in terms of a single, unifying molecular scheme. Now cardiac sodium channels have been analyzed by high-resolution single-channel recordings over a broad range of potentials. Channels exhibited both complex and simple gating patterns at different voltages. Such behavioral diversity can be explained by the balance between two molecular transitions whereby channels can exit the open state.     

Sodium current-induced release of calcium from cardiac sarcoplasmic reticulum

The role of sodium-calcium exchange at the sarcolemma in the release of calcium from cardiac sarcoplasmic reticulum was investigated in voltage-clamped, isolated cardiac myocytes. In the absence of calcium entry through voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores. This process was dependent on sodium entry through tetrodotoxin-sensitive sodium channels. Calcium release under these conditions was also dependent on extracellular calcium concentration, suggesting a calcium-induced trigger release mechanism that involves calcium entry into the cell by sodium-calcium exchange. This sodium current-induced calcium release mechanism may explain, in part, the positive inotropic effects of cardiac glycosides and the negative inotropic effects of a variety of antiarrhythmic drugs that interact with cardiac sodium channels. In response to a transient rise of intracellular sodium, sodium-calcium exchange may promote calcium entry into cardiac cells and trigger sarcoplasmic calcium release during physiologic action potentials.     

Exchange of conduction pathways between two related K+ channels

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.     

桔小实蝇电压门控钠离子通道基因cDNA克隆及其生物信息学分析

【目的】克隆获得桔小实蝇（Bactrocera dorsalis）电压门控钠离子通道基因cDNA序列,明确其典型特征,为研究桔小实蝇抗性分子机理奠定基础。【方法】采用RT-PCR和PCR技术,克隆桔小实蝇钠离子通道基因cDNA序列,利用相关软件对其序列进行生物信息学分析。【结果】克隆得到1条长为6 446 bp的cDNA序列,包含1个6 405 bp的完整开放阅读框,共编码2 134个氨基酸。同源比对发现,桔小实蝇与果蝇（Drosophila melanogaster,NP_001188635）和家蝇（Musca domestica,AAB47604）钠离子通道基因相似度分别高达91.7%和86.9%,而与人的钠离子通道Nav1.2基因（Homo sapiens,NP_066287）相似度为42.3%。所克隆序列包含昆虫钠离子通道所有典型特征。【结论】成功地从桔小实蝇中克隆得到钠离子通道基因完整开放阅读框。该钠离子通道基因存在丰富的选择性剪接,发现了3个可能与抗性相关的碱基取代。钠离子通道基因有可能发展成为一种昆虫系统发育研究的分子标记。     

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.     

Gating currents of the sodium channels: three ways to block them

Preceding the opening of the sodium channels of axon membrane there is a small outward current, gating current, that is probably associated with the molecular rearrangements that open the channels. Gating current is reversibly blocked by three procedures that block the sodium current: (i) internal perfusion with zinc ions, (ii) inactivation of sodium conductance by brief depolarization, and (iii) prolonged depolarization.     

Calcium and sodium channels in spontaneously contracting vascular muscle cells

Electrophysiological recordings of inward currents from whole cells showed that vascular muscle cells have one type of sodium channel and two types of calcium channels. One of the calcium channels, the transient calcium channel, was activated by small depolarizations but then rapidly inactivated. It was equally permeable to calcium and barium and was blocked by cadmium, but not by tetrodotoxin. The other type, the sustained calcium channel, was activated by larger depolarizations, but inactivated very little; it was more permeable to barium than calcium. The sustained calcium channel was more sensitive to block by cadmium than the transient channel, but also was not blocked by tetrodotoxin. The sodium channel inactivated 15 times more rapidly than the transient calcium channel and at more negative voltages. This sodium channel, which is unusual because it is only blocked by a very high (60 microM) tetrodotoxin concentration but not by cadmium, is the first to be characterized in vascular muscle, and together with the two calcium channels, provides a basis for different patterns of excitation in vascular muscles.     

Permeation of calcium ions through non-NMDA glutamate channels in retinal bipolar cells.

The conduction of calcium ions through glutamate-gated channels is important in the induction of long-term potentiation and may trigger other cellular changes. In retinal bipolar cells, which lack the N-methyl-D-aspartate (NMDA) type of glutamate-gated channel, calcium permeability through non-NMDA channels was examined. Changes in extracellular calcium concentration unexpectedly affected the reversal potential for glutamate-induced currents in a manner consistent with these channels being highly permeable to calcium. External magnesium ions promote desensitization of these non-NMDA channels in a voltage-independent way. Thus, in addition to non-NMDA channels that conduct only sodium and potassium, there is a class that is also permeable to calcium.